Oligonucleotide-directed, site-specific mutagenesis is used to dissect the biochemical basis of oncogenic activation and of enzymatic activity of the ras oncogene. Studies are directed toward an understanding of the interrelationship between the known properties of the ras gene product. Mutagenesis of the ras oncogene in specific regions of the protein has been designed to explore the active center which is believed to be responsible for these properties. Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-ras-H oncogene, which substituted amino acid residues within the nucleotide- binding consensus sequence, GXXXXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral ras-H product, p21, lost its GTP-binding and autokinase activities. Other substitutions at position 22, 33, 51 or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into pSV2neo plasmid. Clones with valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. Ras, with a valine mutation at glycine- 10, which had lost its ability to bind GTP and its autokinase activity in vitro and in vivo, was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer p21 its active conformation without participation of ligand binding.